While working as a waitress at a sushi bar in Westmount, a regular customer, a Canadian television producer, overheard Lefevre telling the hostess that she wanted to be an actress.[10] The producer got Lefevre her first audition, for a role in the sitcom Student Bodies. She didn't yet have a head shot, so submitted a Polaroid picture.[11] She didn't land the part but got a call back from the casting director, leading to a role in the Canadian TV series Big Wolf on Campus in 1999, playing Stacey Hanson.[9] Lefevre continued attending McGill between shoots, but never finished a degree.[12][13]

Lefevre played the renegade vampire Victoria Sutherland in the film Twilight (2008), based on the novel of the same name by Stephenie Meyer. Lefevre wrote an impassioned letter to the director, explaining her desire to work with the filmmaker.[15] Lefevre described the essence of her character as "pure evil, pure instinct, pure malice, and very feline".[16] After reading that the author used the word "feline" to describe her character's agility, Lefevre watched lion attacks on YouTube to separate the movements of her character from those of normal people.[17] She also took trapeze classes in preparation for the wire work in the film.[15] Lefevre spent hours working on the costumes for her character, and described herself as "obsessed" with vampires after reading Bram Stoker's Dracula at the age of 14.[18]

Lefevre was, at times, overwhelmed by the scrutiny she received from her involvement with Twilight. She participated in a promotional tour in November 2008, where she met and signed autographs for Twilight fans at Hot Topic stores in the U.S.[19] "It's the closest I will ever come in my life to being a rock star," she told The Canadian Press, describing an appearance on MuchMusic, where over 1,500 fans showed up in Toronto.[20] Over 2,500 fans showed up at a Wal-Mart in Salt Lake City, where Lefevre appeared to promote the DVD release of the film.[21] Overall though, Lefevre described herself as delighted by the attention and excitement of the fans.[20] She appeared in New Moon, the film's sequel, based on Meyer's second novel,[15] which she completed shooting in Vancouver in May 2009.

Lefevre did not reprise her role of Victoria in Eclipse, the third film of the Twilight series, and was replaced by Bryce Dallas Howard. Summit Entertainment, the studio behind the films, attributed the change to scheduling conflicts; Eclipse and Barney's Version, an independent Canadian feature Lefevre had signed on to, both began filming on 17 August 2009.[22] Lefevre responded that she was "stunned" by the decision and "greatly saddened" not to continue her portrayal of Victoria, and never thought she would "lose the role over a 10 day overlap", in a statement to Access Hollywood.[23] The studio responded in a counter-statement, "The Twilight Saga: Eclipse is an ensemble production that has to accommodate the schedules of numerous actors while respecting the established creative vision of the filmmaker and most importantly the story."[24] Lefevre had appeared at the 2009 San Diego Comic-Con to promote New Moon the weekend before she was replaced.[25]

Enthusiastic fans of the series reacted to the news of Lefevre's replacement with online petitions urging her return, and "Bring Back Rachelle" became a top-trending topic on Twitter in the afternoon of 29 July 2009.[24] Lefevre told Extra that she was "absolutely blown away" by support from fans, who also made her a tribute video on YouTube.[26] Lefevre did not attend the Los Angeles premiere of New Moon, tweeting that the event "was just 2 emotional 4 me & I couldn't manage it." (sic)[27]

Lefevre appeared in the pilot episode of the ABC television drama The Deep End on 21 January 2010.[32] She starred on the television drama Off the Map as Dr. Ryan Clark, a young doctor working in a South American medical clinic.[33] Developed by Grey's Anatomy-creator Shonda Rhimes, the series was filmed in Hawaii, debuted on 12 January 2011 and ran for 13 episodes before being canceled.[34] Lefevre was the voice of the 31st annual Genie Awards in March 2011, along with host William Shatner.[35] She was also cast in the NBC pilot, The Crossing, a drama set during the American Civil War. Lefevre plays Anna, a widow who begins an affair with a soldier.[36]